CP27: Expression, Regulation, and Function of a Chromatin Complex Member

CP27 is a novel gene involved in early vertebrate development that features a distinct expression pattern during development. The cp27 gene contains an open reading frame of 295 amino acids corresponding to a predicted molecular mass of 33kDa. Here we have characterized the CP27 promoter and performed a first analysis of CP27 function in development. To study cp27 gene regulation, we have examined its transcriptional activity, and identified transcription factor binding sites in the proximal promoter region. Promoter function analysis of the 5′ flanking region by progressive 5′ deletion mutations localized transcription repression elements. Functional studies indicated two function-cooperative CCAAT boxes and identified the NF-Y transcription factor as the CCAAT activator controlling transactivation of the CP27 promoter. The maintenance and differentiation of ES cells depends on the regulation of gene expression through the coordinated binding of transcription factors to regulatory promoter elements. One of the genes involved in embryonic development is the chromatin factor CP27. Here we report that CP27 gene expression in ES cells is controlled by CCAAT and E-box cis-acting regulatory elements and their corresponding transcription factors NF-Y and USF1. Specifically, USF1 interacts with the E-box of the cp27 proximal promoter and NF-Y interacts with the CCAAT-box. NF-Y and USF1 also interacted with each other and activated the cp27 promoter. Chromatin dynamics assume key functions in the lineage differentiation and gastrulation of bilaterian embryos. Here we demonstrate that a novel chromatin factor CP27 is required for mouse development, germ layer differentiation, and the formation of an archenteron. Loss of CP27 caused severely disturbed epiblast development and prolonged endoderm survival. cp27 Null mice expressed residual levels of the pluripotency factor Nanog and the variant histone H2A.Z. CP27 interacted with H2A.Z on a chromatin level, and a chromatin complex containing both CP27 and H2A.Z differentially bound to the Nanog promoter. Together, these findings suggest a direct link between CP27 and H2A.Z chromatin complex interactions and ES cell lineage specification via Nanog. Together, our data indicate that CP27 is chromatin factor involved in early embryonic development that is regulated by the universal transcription factor NF-Y in combination with the USF-1 enhancer

Long-Lived Luminogenic Probe for Detection of RNA in a Crude Solution of Living Bacterial Cells

A pre-type sensitizer for a lanthanide complex on an oligonucleotide was successfully converted to a perfect final structure in a target DNA/RNA-templated reaction, without any chemical reagent or enzyme, under neutral conditions. The final form of the lanthanide–oligonucleotide provided a long-lived luminescence signal, appropriate for time-gated luminescence analysis and signal amplification. Target DNA/RNA-assisted time-gated luminescence analysis is a powerful tool for elimination of autofluorescence and detection of target RNA in living bacterial cells

Automatic Pseudorotaxane Formation Targeting on Nucleic Acids Using a Pair of Reactive Oligodeoxynucleotides

Here we report a novel method to form a pseudorotaxane architecture using only a pair of reactive oligodeoxyribonucleotides (ODNs), which we designed and synthesized, and then performed the pseudorotaxane formation reaction with both DNA and RNA oligonucleotides. The reaction proceeded smoothly without any extra reagents at 37 °C and pH 7.2, leading to the formation of a stable complex on a denaturing polyacrylamide gel. Interestingly, the pseudorotaxane was formed with the cyclized ODN reversibly by the slipping process. This new pseudorotaxane formation represents a promising method for developing new DNA nanotechnologies and antisense oligonucleotides

Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces

The regeneration of lost periodontal ligament (PDL) and alveolar bone is the purpose of periodontal tissue engineering. The goal of the present study was to assess the suitability of 3 odontogenic progenitor populations from dental pulp, PDL, and dental follicle for periodontal regeneration when exposed to natural and synthetic apatite surface topographies. We demonstrated that PDL progenitors featured higher levels of periostin and scleraxis expression, increased adipogenic and osteogenic differentiation potential, and pronounced elongated cell shapes on barren root chips when compared with dental pulp and dental follicle cells. When evaluating the effect of surface characteristics on PDL progenitors, natural root surfaces resulted in elongated PDL cell shapes, whereas PDL progenitors on synthetic apatite surfaces were rounded or polygonal. In addition, surface coatings affected PDL progenitor gene expression profiles: collagen I coatings enhanced alkaline phosphatase and osteocalcin expression levels and laminin-1 coatings increased epidermal growth factor (EGF), nestin, cadherin 1, and keratin 8 expression. PDL progenitors seeded on natural tooth root surfaces in organ culture formed new periodontal fibers after 3 weeks of culture. Finally, replantation of PDL progenitor-seeded tooth roots into rat alveolar bone sockets resulted in the complete formation of a new PDL and stable reattachment of teeth over a 6-month period. Together, these findings indicate that periodontal progenitor cell type as well as mineral surface topography and molecular environment play crucial roles in the regeneration of true periodontal anchorage

Prepared DNA templates. ProX tag sequence (SKQIEVN–amber-SNE) was contained in PEGx(ProX)–FLAG.

<p>Prepared DNA templates. ProX tag sequence (SKQIEVN–amber-SNE) was contained in PEGx(ProX)–FLAG.</p

Illustration of the synthesis of polyethylene-glycol-carrying tRNA and the incorporation of PEG into a polypeptide by in vitro translation.

<p>Illustration of the synthesis of polyethylene-glycol-carrying tRNA and the incorporation of PEG into a polypeptide by in vitro translation.</p

Western blot analysis using anti-FLAG tag antibody of the in vitro translation of thioredoxin (Trx) with an incorporated PEG chain.

<p>a. Dependence of protein synthesis on PEG–AF–tRNA concentration. b. PEG-incorporated protein synthesis under different conditions.</p

Triphenylphosphinecarboxamide: An Effective Reagent for the Reduction of Azides and Its Application to Nucleic Acid Detection

A series of triphenylphosphinecarboxamide (TPPc) derivatives were designed and synthesized as alternative reagents to triphenylphosphine for the facile reduction of azides. The TPPc derivatives performed as efficient reducing agents for the synthesis of primary amines without the need for an additional hydrolysis procedure. The TPPc derivatives were also applied to nucleic acid sensing using a RhAz-oligonucleotide conjugate in a DNA-templated fluorogenic reaction

Mass spectra of polyethylene-glycol-carrying tRNAs.

<p>The molecular weights of PEG16–tRNA, PEG24–tRNA, and PEG24–tRNA were calculated to be 24485.69, 24838.10, and 25983.45, respectively.</p

Mass spectrometry analysis of poly(ethylene glycol)-incorporated polypeptide.

<p>a. Mass spectra of polypeptide incorporated with poly(ethylene glycol) chain. PEG4–<i>FLAG</i>, calculated 2681.177 for (M+H)⁺, found 2681.152; PEG12–<i>FLAG</i>, calculated 3033.386 for (M+H)⁺, found 3033.972; PEG16–<i>FLAG</i>, calculated 3209.492 for (M+H)⁺, found 3209.529; PEG24–<i>FLAG</i>, calculated 3563.890 for (M+H)⁺, found 3563.712; PEG48–<i>FLAG</i>, calculated 4709.244 for (M+H)⁺, not found. b. Molecular weight dependence of coefficient of incorporation of poly(ethylene glycol) into a polypeptide by in vitro translation.</p